1887

Abstract

Listeriolysin O (LLO, -encoded), a major virulence factor secreted by the bacterial pathogen , is synthesized as a precursor of 529 residues. To impair LLO secretion, the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the mutant LLO protein was expressed in a -negative derivative of . Unexpectedly, the mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic. N-terminal sequencing of the secreted LLO molecule revealed that N-terminal processing of the preprotein occurred three residues downstream of the natural cleavage site. expressing this truncated LLO showed a reduced capacity to disrupt the phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain showed a 100-fold decrease in virulence in the mouse model. These results suggest that the first N-terminal residues of mature LLO participate directly in phagosomal escape and bacterial infection.

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2003-05-01
2020-01-29
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References

  1. Antelmann H., Tjalsma H., Voigt B., Ohlmeier S., Bron S., van Dijl J. M., Hecker M.. 2001; A proteomic view on genome-based signal peptide predictions. Genome Res11:1484–1502
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Smith J. A., Seidman J. G., Struhl K.. 1990; Current Protocols in Molecular Biology New York: Wiley Interscience;
    [Google Scholar]
  3. Berche P.. 1995; Bacteremia is required for invasion of the murine central nervous system by Listeria monocytogenes . Microb Pathog18:323–336
    [Google Scholar]
  4. Bron S., Bolhuis A., Tjalsma H., Holsappel S., Venema G., van Dijl J. M.. 1998; Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli. J Biotechnol64:3–13
    [Google Scholar]
  5. Cosma C. L., Crotwell M. D., Burrows S. Y., Silhavy T. J.. 1998; Folding-based suppression of extracytoplasmic toxicity conferred by processing-defective LamB. J Bacteriol180:3120–3130
    [Google Scholar]
  6. De Chastellier C., Berche P.. 1994; Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect Immun62:543–553
    [Google Scholar]
  7. Decatur A. L., Portnoy D. A.. 2000; A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. Science290:992–995
    [Google Scholar]
  8. Dramsi S., Biswas I., Maguin E., Braun L., Mastroeni P., Cossart P.. 1995; Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigene family. Mol Microbiol16:251–261
    [Google Scholar]
  9. Drevets D. A., Sawyer R. T., Potter T. A., Campbell P. A.. 1995; Listeria monocytogenes infects human endothelial cells by two distinct mechanisms. Infect Immun63:4268–4276
    [Google Scholar]
  10. Dubail I., Berche P., Consortium T. E. L. G., Charbit A.. 2000; Listeriolysin O as a reporter to identify constitutive and in vivo inducible promoters in the pathogen Listeria monocytogenes . Infect Immun68:3242–3250
    [Google Scholar]
  11. Erdenlig S., Ainsworth A. J., Austin F. W.. 1999; Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish. Appl Environ Microbiol65:2827–2832
    [Google Scholar]
  12. Gaillard J. L., Berche P., Sansonetti P.. 1986; Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes . Infect Immun52:50–55
    [Google Scholar]
  13. Gaillard J. L., Berche P., Mounier J., Richard S., Sansonetti P.. 1987; In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun55:2822–2829
    [Google Scholar]
  14. Gaillard J. L., Jaubert F., Berche P.. 1996; The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. J Exp Med183:359–369
    [Google Scholar]
  15. Garandeau C., Reglier-Poupet H., Dubail I., Beretti J.L., Berche P., Charbit A.. 2002; The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence. Infect Immun70:1382–1390
    [Google Scholar]
  16. Geoffroy C., Gaillard J. L., Alouf J. E., Berche P.. 1989; Production of thiol-dependent haemolysins by Listeria monocytogenes and related species. J Gen Microbiol135:481–487
    [Google Scholar]
  17. Glaser P., Frangeul L., Buchrieser C.. 51 other authors 2001; Comparative genomics of Listeria species. Science294:849–852
    [Google Scholar]
  18. Guzman C. A., Rohde M., Chakraborty T., Domann E., Hudel M., Wehland J., Timmis K. N.. 1995; Interaction of Listeria monocytogenes with mouse dendritic cells. Infect Immun63:3665–3673
    [Google Scholar]
  19. Jones S., Portnoy D. A.. 1994; Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect Immun62:5608–5613
    [Google Scholar]
  20. Kathariou S., Metz P., Hof H., Goebel W.. 1987; Tn 916 -induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes . J Bacteriol169:1291–1297
    [Google Scholar]
  21. Kreft J., Funke D., Haas A., Lottspeich F., Goebel W.. 1989; Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b. FEMS Microbiol Lett48:197–202
    [Google Scholar]
  22. Kuhn M., Goebel W.. 1989; Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect Immun57:55–61
    [Google Scholar]
  23. Lety M. A., Frehel C., Dubail I., Beretti J. L., Kayal S., Berche P., Charbit A.. 2001; Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence of Listeria monocytogenes . Mol Microbiol39:1124–1140
    [Google Scholar]
  24. Lety M. A., Frehel C., Berche P., Charbit A.. 2002; Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes . Mol Microbiol46:367–379
    [Google Scholar]
  25. Mackaness G. B.. 1962; Cellular resistance to infection. J Exp Med116:381–406
    [Google Scholar]
  26. Mengaud J., Vicente M. F., Chenevert J., Pereira J. M., Geoffroy C., Gicquel-Sanzey B., Baquero F., Perez-Diaz J. C., Cossart P.. 1988; Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes . Infect Immun56:766–772
    [Google Scholar]
  27. Paetzel M., Dalbey R. E., Strynadka N. C.. 2000; The structure and mechanism of bacterial type I signal peptidases. A novel antibiotic target. Pharmacol Ther87:27–49
    [Google Scholar]
  28. Pamer E. G., Sijts A. J., Villanueva M. S., Busch D. H., Vijh S.. 1997; MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses. Immunol Rev158:129–136
    [Google Scholar]
  29. Portnoy D. A., Jacks P. S., Hinrichs D. J.. 1988; Role of hemolysin for the intracellular growth of Listeria monocytogenes . J Exp Med167:1459–1471
    [Google Scholar]
  30. Pugsley T.. 1993; The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev57:50–108
    [Google Scholar]
  31. Raivio T. L., Laird M. W., Joly J. C., Silhavy T. J.. 2000; Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response. Mol Microbiol37:1186–1197
    [Google Scholar]
  32. Rechsteiner M., Rogers S. W.. 1996; PEST sequences and regulation by proteolysis. Trends Biochem Sci21:267–271
    [Google Scholar]
  33. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Expression of cloned genes in Escherichia coli . In Molecular Cloning: a Laboratory Manual pp17.37–17.41 Edited by Nolan C. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Shen L. M., Lee J. I., Cheng S. Y., Jutte H., Kuhn A., Dalbey R. E.. 1991; Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase. Biochemistry30:11775–11781
    [Google Scholar]
  35. Trieu-Cuot P., Carlier C., Poyart-Salmeron C., Courvalin P.. 1990; A pair of mobilizable shuttle vectors conferring resistance to spectinomycin for molecular cloning in Escherichia coli and in Gram-positive bacteria. Nucleic Acids Res18:4296
    [Google Scholar]
  36. Van Weyly K. H. M., Swaving J., Freudl R., Driessen A. J. M.. 2001; Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev25:437–454
    [Google Scholar]
  37. Varshavsky A.. 1996; The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci U S A93:12142–12149
    [Google Scholar]
  38. Villanueva M. S., Sijts A. J., Pamer E. G.. 1995; Listeriolysin is processed efficiently into an MHC class I-associated epitope in Listeria monocytogenes -infected cells. J Immunol155:5227–5233
    [Google Scholar]
  39. von Heijne G.. 1990; The signal peptide. J Membr Biol115:195–201
    [Google Scholar]
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